Application of single-cell RNA sequencing methodologies in understanding haematopoiesis and immunology.

The blood and immune system are characterised by utmost diversity in its cellular components. This heterogeneity can solely be resolved with the application of single-cell technologies that enable precise examination of cell-to-cell variation. Single-cell transcriptomics is continuously pushing forward our understanding of processes driving haematopoiesis and immune responses in physiological settings as well as in disease. Remarkably, in the last five years, a number of studies involving single-cell RNA sequencing (scRNA-seq) allowed the discovery of new immune cell types and revealed that haematopoiesis is a continuous rather than a stepwise process, thus challenging the classical haematopoietic lineage tree model. This review summarises the most recent studies which applied scRNA-seq to answer outstanding questions in the fields of haematology and immunology and discusses the present challenges and future directions.The study was supported by Cancer Research UK grant number C45041/A14953 and European Research Council project 677501 – ZF_Bloo

Predictors of chronic fatigue syndrome and mood disturbance after acute infection

Prospective cohort studies following individuals from acute infections have documented a prevalent post-infective fatigue state meeting diagnostic criteria for chronic fatigue syndrome (CFS) – that is, a post-infective fatigue syndrome (PIFS). The Dubbo Infection Outcomes Study (DIOS) was a prospective cohort following individuals from acute infection with Epstein-Barr virus (EBV), Ross River virus (RRV), or Q fever through to assessment of caseness for CFS designated by physician and psychiatrist assessments at 6 months. Previous studies in DIOS have revealed that functional genetic polymorphisms in both immunological (pro- and anti-inflammatory cytokines) and neurological (the purinergic receptor, P2X7) genes are associated with both the severity of the acute infection and subsequent prolonged illness. Principal components analysis was applied to self-report data from DIOS to describe the severity and course of both the overall illness and concurrent mood disturbance. Associations between demographics and acute infection characteristics, with prolonged illness course as well as the PIFS outcome were examined using multivariable statistics. Genetic haplotype-driven functional variations in the neuropeptide Y (NPY) gene previously shown to be associated with brain responses to stress, and to trait anxiety were also examined as predictors. The sample included 484 subjects (51% female, median age 32, IQR 19–44), of whom 90 (19%) met diagnostic criteria for CFS at 6 months. Participants with greater overall illness severity and concurrent mood disturbance in the acute illness had a more prolonged illness severity (HR = 0.39, 95% CI: 0.34–0.46, p 0.05). Severe acute infective illnesses predicted prolonged illness, prolonged mood disturbance and PIFS. These factors may facilitate early intervention to manage both PIFS and mood disturbances

The efficacy of the thyroid peroxidase marker for distinguishing follicular thyroid carcinoma from follicular adenoma

Aim: Expression of thyroid peroxidase (TPO) in the thyroid gland tissue is well known as a sensitive marker of the thyroid malignancy. We have evaluated immunohistochemical assay of TPO for distinguishing follicular thyroid carcinoma from follicular adenoma. Materials and Methods: Sections of formalin-fixed tissues obtained from 92 patients with thyroid tumors (52 follicular carcinomas and 40 follicular adenomas including the Hurthle cell type) were analyzed using a monoclonal antibody (TPO mAb 47) and the avidin-biotin peroxidase complex immunohistochemical technique. Lesions with staining of more than 80% of the follicular cells/specimen were considered benign, while less than 80% were considered malignant. Results: TPO immunostaining correlated with the histopathological diagnosis in 24/40 cases of follicular adenomas and 41/52 cases of follicular carcinomas, giving a specificity of 60% and a sensitivity of 79%. Conclusion: These results suggest that immunohistochemical assay of TPO expression has limited value for the differential diagnosis of follicular thyroid carcinoma from thyroid follicular adenoma.Цель: уровень экспрессии тироидной пероксидазы (ТПО) в ткани щитовидной железы является чувствительным маркером малигнизации этого органа. В работе представлено попытку оценки метода иммуногистохимический детекции ТПО для дифференциальной диагностики фолликулярной карциномы и фолликулярной аденомы щитовидной железы (ФКЩЖ и ФАЩЖ соответственно). Материалы и методы: срезы ткани, зафиксированные в формалине, были получены у 92 пациентов с опухолями щитовидной железы (52 случая — ФКЩЖ и 40 — ФАЩЖ, в том числе тип с клетками Хюртля). Для иммуногистохимического анализа этих срезов использовали моноклональные антитела против ТПО (ТРОmAb47) и авидин-биотиновый комплекс. Препараты опухолей, содержащих более 80% позитивно окрашенных фолликулярных клеток, признавали доброкачественными, а те, что содержали менее 80% таких клеток, — злокачественными. Результаты: интенсивность иммуноокрашивания препаратов коррелировала с гистопатологическим диагнозом в 24 из 40 случаев ФАЩЖ и в 41 из 52 случаев ФКЩЖ. При этом чувствительность метода составляла 79%, специфичность — 60%. Выводы: иммуногистохимический анализ ТПО имеет недостаточную специфичность для дифференциальной диагностики фолликулярной карциномы и фолликулярной аденомы

Choreography, controversy and child sex abuse: Theoretical reflections on a cultural criminological analysis of dance in a pop music video

This article was inspired by the controversy over claims of ‘pedophilia!!!!’ undertones and the ‘triggering’ of memories of childhood sexual abuse in some viewers by the dance performance featured in the music video for Sia’s ‘Elastic Heart’ (2015). The case is presented for acknowledging the hidden and/or overlooked presence of dance in social scientific theory and cultural studies and how these can enhance and advance cultural criminological research. Examples of how these insights have been used within other disciplinary frameworks to analyse and address child sex crime and sexual trauma are provided, and the argument is made that popular cultural texts such as dance in pop music videos should be regarded as significant in analysing and tracing public perceptions and epistemologies of crimes such as child sex abuse

Integrative Single-Cell RNA-Seq and ATAC-Seq Analysis of Human Developmental Hematopoiesis.

Regulation of hematopoiesis during human development remains poorly defined. Here we applied single-cell RNA sequencing (scRNA-seq) and single-cell assay for transposase-accessible chromatin sequencing (scATAC-seq) to over 8,000 human immunophenotypic blood cells from fetal liver and bone marrow. We inferred their differentiation trajectory and identified three highly proliferative oligopotent progenitor populations downstream of hematopoietic stem cells (HSCs)/multipotent progenitors (MPPs). Along this trajectory, we observed opposing patterns of chromatin accessibility and differentiation that coincided with dynamic changes in the activity of distinct lineage-specific transcription factors. Integrative analysis of chromatin accessibility and gene expression revealed extensive epigenetic but not transcriptional priming of HSCs/MPPs prior to their lineage commitment. Finally, we refined and functionally validated the sorting strategy for the HSCs/MPPs and achieved around 90% enrichment. Our study provides a useful framework for future investigation of human developmental hematopoiesis in the context of blood pathologies and regenerative medicine.The study was supported by European Research Council project 677501 – ZF_Blood (to A.C. and A.M.R), EMBO small grant (to A.C.) and a core support grant from the Wellcome Trust and MRC to the Wellcome Trust – Medical Research Council Cambridge Stem Cell Institute

Chemotaxis: a feedback-based computational model robustly predicts multiple aspects of real cell behaviour

The mechanism of eukaryotic chemotaxis remains unclear despite intensive study. The most frequently described mechanism acts through attractants causing actin polymerization, in turn leading to pseudopod formation and cell movement. We recently proposed an alternative mechanism, supported by several lines of data, in which pseudopods are made by a self-generated cycle. If chemoattractants are present, they modulate the cycle rather than directly causing actin polymerization. The aim of this work is to test the explanatory and predictive powers of such pseudopod-based models to predict the complex behaviour of cells in chemotaxis. We have now tested the effectiveness of this mechanism using a computational model of cell movement and chemotaxis based on pseudopod autocatalysis. The model reproduces a surprisingly wide range of existing data about cell movement and chemotaxis. It simulates cell polarization and persistence without stimuli and selection of accurate pseudopods when chemoattractant gradients are present. It predicts both bias of pseudopod position in low chemoattractant gradients and-unexpectedly-lateral pseudopod initiation in high gradients. To test the predictive ability of the model, we looked for untested and novel predictions. One prediction from the model is that the angle between successive pseudopods at the front of the cell will increase in proportion to the difference between the cell's direction and the direction of the gradient. We measured the angles between pseudopods in chemotaxing Dictyostelium cells under different conditions and found the results agreed with the model extremely well. Our model and data together suggest that in rapidly moving cells like Dictyostelium and neutrophils an intrinsic pseudopod cycle lies at the heart of cell motility. This implies that the mechanism behind chemotaxis relies on modification of intrinsic pseudopod behaviour, more than generation of new pseudopods or actin polymerization by chemoattractant

The Negation Bias in Stereotype Maintenance : A Replication in Five Languages

Research on linguistic biases shows that stereotypic expectancies are implicitly reflected in language and thereby subtly communicated to message recipients. Research on the Negation Bias shows that the use of negations (e.g., not stupid vs. smart) is more pronounced in descriptions of stereotype-inconsistent compared with stereotype-consistent behaviors. This article reports a replication study of the original research conducted in Dutch, using newly developed materials, and in five different languages: English, Dutch, Hungarian, Finnish, and Serbian. The results validate the existence of the Negation Bias in all five languages. This suggests that negation use serves a similar stereotype-maintaining function across language families.Peer reviewe

Prevalence and Characteristics Associated With Post-COVID-19 Condition Among Nonhospitalized Adolescents and Young Adults

Importance: The prevalence and baseline risk factors of post-COVID-19 condition (PCC) remain unresolved among the large number of young people who experienced mild COVID-19. Objectives: To determine the point prevalence of PCC 6 months after the acute infection, to determine the risk of development of PCC adjusted for possible confounders, and to explore a broad range of potential risk factors. Design, Setting, and Participants: This cohort study included nonhospitalized individuals from 2 counties in Norway between ages 12 and 25 years who underwent reverse transcription-polymerase chain reaction (RT-PCR) testing. At the early convalescent stage and at 6-month follow-up, participants underwent a clinical examination; pulmonary, cardiac, and cognitive functional testing; immunological and organ injury biomarker analyses; and completion of a questionnaire. Participants were classified according to the World Health Organization case definition of PCC at follow-up. Association analyses of 78 potential risk factors were performed. Exposures: SARS-CoV-2 infection. Main Outcomes and Measures: The point prevalence of PCC 6 months after RT-PCR testing in the SARS-CoV-2-positive and SARS-CoV-2-negative groups, and the risk difference with corresponding 95% CIs. Results: A total of 404 individuals testing positive for SARS-CoV-2 and 105 individuals testing negative were enrolled (194 male [38.1%]; 102 non-European [20.0%] ethnicity). A total of 22 of the SARS-CoV-2-positive and 4 of the SARS-CoV-2-negative individuals were lost to follow-up, and 16 SARS-CoV-2-negative individuals were excluded due to SARS-CoV-2 infection in the observational period. Hence, 382 SARS-CoV-2-positive participants (mean [SD] age, 18.0 [3.7] years; 152 male [39.8%]) and 85 SARS-CoV-2-negative participants (mean [SD] age, 17.7 [3.2] years; 31 male [36.5%]) could be evaluated. The point prevalence of PCC at 6 months was 48.5% in the SARS-CoV-2-positive group and 47.1% in the control group (risk difference, 1.5%; 95% CI, -10.2% to 13.1%). SARS-CoV-2 positivity was not associated with the development of PCC (relative risk [RR], 1.06; 95% CI, 0.83 to 1.37; final multivariable model utilizing modified Poisson regression). The main risk factor for PCC was symptom severity at baseline (RR, 1.41; 95% CI, 1.27-1.56). Low physical activity (RR, 0.96; 95% CI, 0.92-1.00) and loneliness (RR, 1.01; 95% CI, 1.00-1.02) were also associated, while biological markers were not. Symptom severity correlated with personality traits. Conclusions and Relevance: The persistent symptoms and disability that characterize PCC are associated with factors other than SARS-CoV-2 infection, including psychosocial factors. This finding raises questions about the utility of the World Health Organization case definition and has implications for the planning of health care services as well as for further research on PCC

Analysis of endothelial-to-haematopoietic transition at the single cell level identifies cell cycle regulation as a driver of differentiation

Funder: INTENS EU fp8 consortiumFunder: ERC advanced grant New-CholAbstract: Background: Haematopoietic stem cells (HSCs) first arise during development in the aorta-gonad-mesonephros (AGM) region of the embryo from a population of haemogenic endothelial cells which undergo endothelial-to-haematopoietic transition (EHT). Despite the progress achieved in recent years, the molecular mechanisms driving EHT are still poorly understood, especially in human where the AGM region is not easily accessible. Results: In this study, we take advantage of a human pluripotent stem cell (hPSC) differentiation system and single-cell transcriptomics to recapitulate EHT in vitro and uncover mechanisms by which the haemogenic endothelium generates early haematopoietic cells. We show that most of the endothelial cells reside in a quiescent state and progress to the haematopoietic fate within a defined time window, within which they need to re-enter into the cell cycle. If cell cycle is blocked, haemogenic endothelial cells lose their EHT potential and adopt a non-haemogenic identity. Furthermore, we demonstrate that CDK4/6 and CDK1 play a key role not only in the transition but also in allowing haematopoietic progenitors to establish their full differentiation potential. Conclusion: We propose a direct link between the molecular machineries that control cell cycle progression and EHT

Single-cell transcriptional analysis reveals ILC-like cells in zebrafish.

Innate lymphoid cells (ILCs) are important mediators of the immune response and homeostasis in barrier tissues of mammals. However, the existence and function of ILCs in other vertebrates are poorly understood. Here, we use single-cell RNA sequencing to generate a comprehensive atlas of zebrafish lymphocytes during tissue homeostasis and after immune challenge. We profiled 14,080 individual cells from the gut of wild-type zebrafish, as well as of rag1-deficient zebrafish that lack T and B cells, and discovered populations of ILC-like cells. We uncovered a rorc-positive subset of ILCs that could express cytokines associated with type 1, 2, and 3 responses upon immune challenge. Specifically, these ILC-like cells expressed il22 and tnfa after exposure to inactivated bacteria or il13 after exposure to helminth extract. Cytokine-producing ILC-like cells express a specific repertoire of novel immune-type receptors, likely involved in recognition of environmental cues. We identified additional novel markers of zebrafish ILCs and generated a cloud repository for their in-depth exploration.The study was supported by Cancer Research UK grant number C45041/A14953 (to A.C. and E.I.A.), European Research Council project 677501 – ZF_Blood (to A.C. and P.M.S.), EMBO Long-Term Fellowship ALTF-807-2015 (to P.P.H), ANR grant 17-CE15-0017-01 – ZF-ILC (to P.P.H) and ANR-16-CE20-0002-03 (to J.-P.L), H2020-MSCA-IF-2015 grant 708128 – ZF-ILC (to P.P.H), ANR-10-LABX-73 (‘revive’ to P. Herbomel) and a core support grant from the Wellcome Trust and MRC to the Wellcome Trust – Medical Research Council Cambridge Stem Cell Institute